The signaling achieved by Sonic hedgehog (Shh) plays an essential role in vertebrate development and in the maintenance of several postembryonic stem cell populations. An obligatory component of the pathway utilized by Shh is Smoothened, a 7-transmembrane protein. We have determined using direct assays of G protein activation that mouse Smoothened couples unequivocally to all members of the Gi family of heterotrimeric G proteins. We have also demonstrated that pertussis toxin, which disrupts coupling of Gi to receptors, inhibits the activation of Gli transcription factors by Shh in NIH 3T3 fibroblasts, a commonly employed model of Shh action. The overall goal of the proposed studies is to understand better the mechanisms of transduction relevant to Shh signaling, with an emphasis on those mediated through, or in conjunction with, G proteins. The first aim is to evaluate the selectivity of Smoothened for G proteins within and beyond the Gi family. We will follow up differences in strength of coupling to the most important members of the Gi family and reports that Smo can couple to members of the G12 family as well. The second aim is to identify the target(s) for Gi relevant to the actions of Shh. We will evaluate pertussis toxin-sensitive regulation by Shh of suspected targets, mimic the actions of targets in pertussis toxin-treated cells, and evaluate recruitment through Gi of 2-arrestins. The third aim is to evaluate the requirement for Gi among primary cells serving as targets for Shh, specifically primary fibroblasts, cerebellar granule precursors, and neocortical oligodendrocyte progenitors. The fourth specific aim is to determine what signaling is required by Smo beyond the activity of Gi. We are interested in testing whether Gi and 2-arrestins are both required and together sufficient for activation of Gli transcription factors. Sonic hedgehog is a protein that plays an essential role in embryonic development and tissue regeneration. Deficits in signaling by this protein lead to developmental defects, while unrepressed signaling leads to several cancers. An understanding of the mechanisms by which Sonic hedgehog signals will provide important leads into how that signaling might be manipulated in a therapeutic context.